Background
Bladder urothelial carcinoma (BLCA) is the most common neoplasm in the urological system in China [
1]. Five to 10 % of patients are already at metastatic stage at diagnosis, and around 50% of patients will develop local or distant disease progression after radical cystectomy [
2]. Conventional pathological predictors such as tumor stage and grade are generally inadequate and often inaccurate to predict the heterogeneous behavior of BLCA [
3]. Identification of recurrent genetic alteration in BLCA is critical for discovering genes that drive BLCA and identifying prognostic biomarkers for stratification of patients with different risks of prognosis [
4]. With the advent of next-generation sequencing technology and computational biology, the understanding of BLCA has entered a new era [
4]. Gaining insight into the biology of bladder cancer may reveal numerous biomarkers that enhance the sensitivity and specificity of early diagnosis and treatment of BLCA [
5].
Topoisomerase-II alpha (TOP2A) is an essential nuclear enzyme which regulates the topological state of DNA during transcription and is involved in the processes of chromosome condensation and chromatid separation [
6]. Several studies have reported that higher expression levels of TOP2A are indicative of poor prognosis in a variety of human cancers [
6‐
9]. Moreover, TOP2A is the target for some of the most widely used chemotherapeutic drugs for treatment of human cancers [
10,
11]. Despite the well-known association between TOP2A expression and aggressiveness of different cancers, the underlying role of TOP2A in the BLCA remains unclear.
In the present study, we explored the expression and function of TOP2A in BLCA patient samples and cell lines. We found that TOP2A was overexpressed in BLCA, and higher TOP2A expression was associated with poorer cancer-specific, progression-free and recurrence-free survival. We also revealed that TOP2A regulated proliferation and invasion, and played a role associated with anti-apoptosis in BLCA.
Methods
Clinical samples and cell lines
Ten pairs of BLCA samples (five muscle-invasive bladder cancer (MIBC) and five non-muscle invasive bladder cancer (NMIBC), eight high-grade and two low-grade) and their matched adjacent normal epithelial tissue were collected for RNA-sequencing (RNA-seq) from patients who were treated with cystectomy from June 2013 to May 2014. Another 40 BLCA samples (21 MIBC and 19 NMIBC, 26 high-grade and 14 low-grade) were collected from transurethral resection of bladder cancer (TURBT) or cystectomy for further validation by real time quantitative polymerase chain reaction (RT-qPCR). A total of 209 formalin-fixed BLCA tumors (130 MIBC and 79 NMIBC, 158 high-grade and 51 low-grade, median age 69 years, 144 males and 65 females, 144 from cystectomy and 65 from TURBT) were collected for immunohistochemistry from July 2008 to December 2015. No patient received chemotherapy or radiotherapy previously in this study. All clinical samples were obtained after written informed consents were provided by the patients, and the study protocol was approved by the Ethics Committee of Changhai Hospital. BLCA cells lines used in this study were purchased from the American Type Culture Collection (Manassas, VA, U.S.) and cells were cultured with the recommended medium and condition. Gemcitabine (Catalog No.S1149) and doxorubicin (Catalog No.S1208) was purchased from Selleck.
Gene expression analysis by RNA-sequencing
RNAs were purified from tumor tissue for RNA-Seq analysis by Beijing Genomics Institute (Shenzhen, China). All samples were examined by pathologists to ensure that tumor sample had tumor density > 80% and the adjacent normal tissue were without tumor contamination. Experimental procedures of RNA-Seq were described previously [
12,
13].
Database mining
Public databases: gene expression omnibus (GEO, GSE31684,
n = 93) (
http://www.ncbi.nlm.nih.gov/gds/), and two comprehensive studies, i.e. The Cancer Genome Atlas (TCGA, bladder cancer,
n = 413) and MSKCC (bladder cancer,
n = 97) on BLCA gene expression profile and prognosis in cBioPortal (
http://www.cbioportal.org/) were included for data mining.
RNA extraction and RT-qPCR
Total RNA was extracted by TRIzol reagent (Invitrogen™), and the cDNA was synthesized using a PrimeScript RT Reagent kit (Takara, Japan) following the manufacturer’s instruction. RT-qPCR was conducted on an Applied Biosystems Step One Plus (Agilent, USA) using SYBR® Premix Ex Taq™ (Takara, Japan). The relative fold changes were calculated with 2 − ΔΔCT methods. The primers used are as follows: 5′-CATTGAAGACGCTTCGTTATGG-3′ (forward) and 5′- CCAGTTGTGATGGATAAAATTAATCAG-3′ (reverse) for TOP2A gene; 5′-ACGGTAGCTGCTAAAAAGGGAA-3′ (forward) and 5′- GGATGTTTTCCTGCCAGGGT-3′ (reverse) for TOP2B; 5′- ACCACAGTCCATGCCATCAC-3′ (forward), 5′- TCCACCACCCTGTTGCTGTA-3′ (reverse) for GAPDH.
Immunohistochemistry
Formalin-fixed paraffin-embedded BLCA samples were cut into 5-μm-thick sections. Primary antibody (1:300) anti-TOP2A (ab52934) and anti-Ki67 (ab15580) was purchased from Abcam®. Immunohistochemistry staining was conducted according to the instruction of the immunohistochemistry kit (BioGenex, Fremont, CA, U.S.). The immunohistochemical stain of TOP2A was evaluated by pathologists in a blinded fashion and was scored on the percentage of positive tumor cell nucleus (negative, score 0; < 1/3, score 1; 1/3–2/3, score 2; > 2/3, score 3). Scores of 0–1 were defined as low expression, and 2–3 were defined as high expression.
RNA interference and lentivirus construction
The small interference RNA (siRNA) against TOP2A (5′- GGUCAGAAGAGCAUAUGAUTT-3′; antisense, 5′- AUCAUAUGCUCUUCUGACCTT-3′ for siRNA-1; sense, 5′-GACCAACCUUCAACUAUCUTT-3′; antisense, 5′- AGAUAGUUGAAGGUUGGUCTT-3′ for siRNA-2; sense, 5′-GCTGCGGACAACAAACAAATT − 3′; antisense, 5′- GCTATCAGCCTGGCCTTTATT-3′ for siRNA-3) and non-specific siRNA (sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT − 3′) were purchased from GenePharma, Shanghai, China. Cells were transfected with siRNA according to the instruction of the manufacturer (Lipofectamine® RNAiMAX, Invitrogen). Lentivirus with small hairpin RNA against TOP2A (5′- GATCCGGTCAGAAGAGCATATGATTTCAAGAGAATCATATGCTCTTCTGACCTTTTTTC-3′) and non-specific small hairpin RNA (5′- CTAGCCCGGCCAAGGAAGTGCAATTGCATACTCGAGTATGCAATTGCACTTCCTTGGTTTTTTGTTAAT-3′) were purchased from Hanbio (Shanghai, China). J82 Cells were transfected with lentiviral shRNA at a multiplicity of infection of about 30 in the presence of 5 μg/ml polybrene. Puromycin was added to medium at the concentration of 2.5 mg/ml for stable cell line selection.
Cell proliferation, wound-healing and Matrigel invasion assays
Cell proliferation and drug sensitivity assay was conducted per the instruction of Cell Counting Kit-8 (CCK-8; Dojindo). Cells with a density of 3 × 103 per well were seeded in triplicates in 96-well plates with 200 μL medium, and cell viability was determined using CCK-8 kit by adding 20 μL reagent. The cells were incubated for another 2 h and then the absorbance at 495 nm was measured with EnSpire Reader. Wound-healing assay was conducted by seeding cells in triplicate in 6-well plates. After the cell density reached about 80%, then 10 μL pipette tips were applied to create a cell-free trip area and the floating cells were washed away by PBS buffer. Cells were then cultured with pure medium without 10% fetal bovine serum (FBS). Photographs of the scratched area were taken at 0 h, 12 h, 24 h and 72 h, respectively. Invasion assays were performed with the transwell chamber with Matrigel (Millipore, USA). Cells with a density of 5 × 104 were seeded into the upper chamber and cultured with 500 μL pure medium without 10% FBS, and the lower chamber were added with 1 ml complete medium. After 24 h or 48 h, cells remaining on the upper membrane were removed with cotton wool, and then placed into 4% formalin for 20 min. The cells on the membrane were stained with 0.1% crystal violet for 30 min. Microscope photographs of 10 random filed of the membrane were taken, and cells were counted for further statistical analyses.
All animal experiments were conducted with the approval of the Scientific Investigation Board of the Second Military Medical University. J82 cells infected with lentivirus against TOP2A and non-specific lentivirus were injected subcutaneously into the lower flank of female athymic BALB/c nude mice. Each group had 6 mice and each mouse was injected with 1*106 J82 cells. Tumor size was measured weekly and tumor volume was calculated using the following formula: Volume = Length x width2x 0.5(mm3). Mice with tumor diameter > 1.5 cm or with losses in body weight of > 20% were promptly euthanized by asphyxiation by CO2 inhalation from compressed gas source. All animal studies were performed according to the Guidelines of the Second Military Medical University for the Care and Use of Laboratory Animals.
Flow cytometry analysis
Cells were collected for flow cytometry analysis 72 h after transfection with TOP2A siRNA or control siRNA. Cells were stained using cell cycle kit (Lianke Biotech Co., Ltd., China) per manufacturer’s instructions, and then analyzed on a Beckman Coulter Analyzer (Beckman Coulter, Brea, CA, USA). Cell cycle data were analyzed with Modfit software. Apoptosis was performed using Annexin V fluorescein isothiocyanate and propidium iodide apoptosis kit (Lianke Biotech Co., Ltd., China). Stained cells were analyzed on a MACSQuant Analyzer (Teterow, Germany), and data was then analyzed by FlowJo v7.6.1 software.
Statistics
At least three independent experiments were performed for each experiment described in this study. Student t test or one-way ANOVA was used to compare continuous parametric data between two groups and multiple groups, respectively. Kaplan-Meier survival curve and log-rank test were applied for survival analysis. Univariable and multivariable Cox proportional hazards models were performed to identify independent risk factors for the prognosis of BLCA. A backward step-down wald selection method was used (the entry with P < 0.05 and removal criteria with P < 0.10). The statistical analysis was performed either by SPSS 19.0 (IBM Inc.) or GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA).
Discussion
Two types of topoisomerase II enzymes were expressed in mammalian cells, TOP2A and TOP2B, but only TOP2A plays a crucial role for cellular viability [
10,
16]. TOP2A gene, mapped to chromosome 17q12-q21, encodes an enzyme which implicates in almost any processes of DNA metabolism including replication, transcription and chromosome segregation during interphase and mitosis [
17,
18]. TOP2A is thus known to be a marker of cell proliferation in normal tissue, and increased TOP2A expression levels are observed in cancer cells compared with non-malignant cells [
19,
20]. Previous studies also suggested that high TOP2A expression levels could indicate tumor aggressiveness and poor outcome [
3,
6,
8,
21,
22].
In the present study, our RNA-Seq data of ten pairs of BLCA samples identified TOP2A gene were significantly upregulated in the tumor samples compared with the matched normal epithelial tissue. We further validated that the expression levels of TOP2A mRNA were elevated in tumors by RT-qPCR in a cohort of 40 samples. We found that TOP2A levels were higher in patients with MIBC and high-grade tumors compared with NMIBC and low-grade tumors, respectively. The results were in line with previous studies that TOP2A expression levels reflected the aggressiveness of tumors [
3,
6,
8,
21,
22]. We further demonstrated that there were significant differences between patients with low- and high-expression of TOP2A protein in terms of CSS, PFS and RFS. However, we failed to find TOP2A as an independent risk factor for prognosis in the multivariable COX regression model, and tumor stage and lymph node status served as the most important prognostic factors for BLCA patients. It indicated that TOP2A was an important but not exclusive contributing factor for the progression of BLCA. This result also agreed with several previous studies in BLCA. Simon et al. [
23] reported that TOP2A and HER-2 expression was co-amplified in BLCA, and TOP2A amplification were associated with advanced tumor stage and high grade, but they also found no independent prognostic value of TOP2A amplification or protein expression in multivariable COX regression analysis. Koren et al. [
24] investigated the expression of TOP2A protein by immunohistochemistry in 57 specimens and found higher TOP2A expression indicated greater probability of BLCA recurrence and lower overall survival. Subgroup analysis of the prognostic value of TOP2A in MIBC and NMIBC in the present study further revealed that TOP2A had better prognostic value for patients with MIBC rather than NMIBC. This was in contrast to a previous study, Nakopoulou et al. [
25] reported that higher TOP2A expression was also indicative of worse prognosis for patients with superficial bladder tumors (
n = 51). Taken together, TOP2A is a promising biomarker for differentiating patients with different risks of BLCA, and combining TOP2A and conventional prognostic factors, such as tumor stage, may increase the accuracy of predicting prognostic outcomes and provide additional arguments for treatment and surveillance decisions.
Whether the level of TOP2A mRNA could serve as a predictor for the prognosis of BLCA is in controversy. In the present study, public databases analysis showed that patients with higher TOP2A mRNA expression did not significantly correlate with the prognosis. We also found no correlation between the level of TOP2A mRNA and protein. It suggests that further studies are warranted to explore the post-transcription mechanisms that contribute to interpret the divergent levels between TOP2A mRNA and protein in BLCA. Meanwhile, Ren et al. [
17] revealed high TOP2A expression correlated with worse cancer prognosis, but no relationship between amplification of TOP2A gene and prognosis of cancer in the meta-analysis of 25 studies. In contrast, Kim et al. [
3] reported that elevated expression of TOP2A mRNA was indicative of high rate of recurrence and progression in NMIBC (
n = 103).
Although TOP2A alteration was closely associated with prognosis of BLCA, the oncogenic function of TOP2A in BLCA has not been studied previously. The biological behavior of cancers is generally influenced by two major biological processes: proliferation and invasion [
25]. In the present study, we found that knockdown of TOP2A could significantly inhibit the proliferation of bladder cancer cells and non-cancerous urothelial cells, which revealed the essential role of TOP2A in cell proliferation. In addition, knockdown of TOP2A strongly suppressed the migration and invasion capacity of J82 and 5637 cells. Furthermore, flow cytometry analysis suggested that TOP2A played a role in anti-apoptosis in BLCA. These results strengthened the evidence that TOP2A involved in the progression of BLCA. Jain et al. [
6] reported that TOP2A was overexpressed in adrenocortical carcinoma and might influence tumor progression, as knockdown of TOP2A in adrenocortical carcinoma cells decreased cell proliferation, and invasion. Gobble et al. [
26] found TOP2A was overexpressed in liposarcoma, and knockdown of TOP2A in liposarcoma cell lines reduced proliferation, invasiveness and increased apoptosis. Taken together, TOP2A was proved to play a major role in proliferation and invasion.
Given the essential role of TOP2A in malignant tumors, TOP2A has held the interest of researchers developing targeted anticancer drugs [
11]. Doxorubicin and etoposide are two most clinically active anticancer agents targeting TOP2A in different cancers [
27‐
29]. Previous studies suggested that the levels of TOP2A expression could determine the response of chemotherapeutic drugs targeting this enzyme [
10,
30,
31]. Tumors with low expression of TOP2A had fewer TOP2A mediated DNA strand breaks induced by targeted drugs and was thus less sensitive than tumors with high level of TOP2A [
30]. In the present study, we showed that TOP2A knockdown induced doxorubicin resistance in J82 cells, however, the sensitivity of different bladder cancer cell lines to doxorubicin was not significantly correlated with the expression level of TOP2A. The results implied that the sensitivity of bladder cancer cell lines to doxorubicin might be determined by multiple factors. In addition to TOP2A down-regulation, AbuHammad et al. [
32] revealed that metabolizing genes (specifically CYP1A1 and CYP1A2) and other genes were crucial for cell cycle, apoptosis and DNA repair were involved in the development of doxorubicin resistance to breast cancer cells. As a result, combinational biomarkers rather than sole TOP2A expression level warrants further investigation to predict the sensitivity of BLCA to doxorubicin.
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